There has been a growing interest in recent years in bacterial Ig (immunoglobulin) receptors, molecules that bind to antibodies through a nonimmune mechanism. This binding is not to the antigen recognition site, which is located in the variable portion of the antibody molecule, but to the constant portion of the antibody. The constant region is common to many types of antibodies, thus bacterial Ig receptors can bind to many types of antibodies. This property makes bacterial Ig receptors useful in a number of immunochemical applications.
Bacterial Ig receptors have a number of useful or potentially useful applications, primarily in the detection of antibodies, the purification of antibodies and the treatment of diseases. The detection of antibodies is required in several phases of laboratory research in immunology, including the screening of hybridoma clones for the secretion of specific monoclonal antibodies, the measurement of the immune response of an immunized animal, and the quantitation of antigens by competitive binding assays. Methods for detecting antibodies using bacterial Ig receptors have been found to be more sensitive and less prone to interference and high background signals than other detection methods [Boyle, M.D.P., Biotechniques 2:334-340 (1984)].
Ig receptors also are useful in purifying antibodies to be used in the purification of protein drugs and as therapeutics. Although a number of methods are known, a popular method involves the use of affinity chromatography on columns of immobilized bacterial Ig receptors. This method is preferred because the columns can be reused many times, thus lowering the expense of purification.
A number of potential clinical uses of bacterial Ig receptors are currently under investigation. They include passing plasma over extracorporeal columns of immobilized Ig receptors, then reinfusing the treated plasma. See, for example, Tenan, D.S., et al., N. Eng. J. Med. 305:1195-1200 (1981).
The best known bacterial Ig receptor is Protein A of Staphylococcus aureus, which binds to the constant F.sub.c domain of immunoglobulin IgG. Other bacterial Ig receptors also have been identified. One of these is known as Protein G of Group G streptococci. Although Protein G is analogous to Protein A, Protein G has several important advantages. For example, Protein G binds to all subclasses of human IgG, whereas Protein A does not bind to the IgG3 subclass [Reis, K. J. et al. J. Immunol. 132:3098-3102 (1984)]. Protein G also is specific for IgG and does not cross-react with human antibodies of type IgA and IgM as Protein A does. [Myhre, E. B. and Kronvall, G. "Immunoglobulin Specificities of Defined Types of Streptococcal Ig Receptors" In: Basic Concepts of Streptococci and Streptococcal Diseases; J. E. Holm and P. Christensen, eds.; Redbook, Ltd., Chertsey, Surrey; pp. 209-210 (1983)]. In addition, Protein G binds to certain animal IgGs to which Protein A binds weakly or not at all. These include bovine, bovine, and caprine IgGl and several subclasses of equine IgG (Reis, K. J. et al., supra). Protein G also has been found superior to Protein A in binding to several subclasses of murine monoclonal antibodies [Bjorck, L. and Kronvall, G. J. Immunol. 133:969-974 ( 1984)]. For these reasons, Protein G is likely to become the bacterial Ig receptor of choice in a variety of applications.
Protein G may be obtained for study by purification from Streptococcal strains which naturally produce it. For example, Streptococcal cells have been treated with proteolytic enzymes (e.g., papain or trypsin) to solubilize the Protein G (which is a cell wall protein), followed by known protein purification procedures (e.g., ion exchange chromatography, gel filtration, and affinity chromatography) to further purify the Protein G (European Patent Application, Publication Number 0 131 142).
Given the advantages, uses and potential uses of Protein G and variants thereof, it would be desirable to be able to produce the variants using recombinant DNA methodology and to immobilize the variants for use in chromatography. Accordingly, it is an object of the present invention to construct and clone genes encoding Protein G and variants thereof and to produce Protein G and variants thereof by transforming a microbial host with the cloned gene and cultivating the host under Protein G-producing conditions. It is also an object of the present invention to immobilize Protein G and variants thereof on solid supports and to use the immobilized variants in chromatographic separation processes.